The invention concerns compositions which comprise colloidal particles on the surface of which biomolecules are adsorbed.
Conjugates of biomolecules, such as proteins or nucleic acids, and colloidal particles are widely used for example as signal transmitters or/and as capture reagents in diagnostic and therapeutic methods. They serve for example as markers in detection procedures such as immunoassays or as microprojectiles for gene transfer. Particles that can be used are particles of metals and metal compounds such as metal oxides, metal hydroxides, metal salts and polymer cores which are coated with metals or metal compounds (cf. e.g. U.S. Pat. No. 4,313,734; Leuvering et al., J. Immunoassay 1 (1980), 77-91; Leuvering Dissertation (1984), Sol Particle Immunoassay (SPIA): The use of Antibody Coated Particles as Labelled Antibodies in Various Types of Immunoassay; Uda et al., Anal. Biochem. 218 (1994), 259-264, DE-OS 41 32 133, page 3, lines 16-18 for applications as a marker and Tang et al., Nature 356 (1992), 152-154; Eisenbraun et al., DNA and Cell Biology 12 (1993), 791-797; Williams et al., Proc. Natl. Acad. Sci. USA 88 (1991), 2726-2730 for gene transfer applications). Furthermore it is also known that non-metallic colloidal particles such as carbon particles can be used (van Amerongen, Anabiotic ""92 (1993), 193-199). At present colloidal gold particles are most frequently used.
Biomolecule-gold conjugates are prepared by firstly preparing gold sols by generally known procedures by reducing tetrachloroauric acid. Subsequently the gold sols are loaded with the biomolecule desired in each case e.g. proteins such as antibodies, protein A, protein G, streptavidin etc. The respective loading conditions (pH, buffer, concentration of the biomolecules etc) depend on the isoelectric point of the biomolecules, the MPA (minimal protecting amount) or/and the specific application of the conjugate (cf. e.g. De Mey, The Preparation and Use of Gold Probes, in: Immunocytochemistry, publisher: J. M. Polak and S.V. Noorden, pages 115-145, Wright, Bristol 1986; J. E. Beesley, Colloidal Gold: A New Perspective for Cytochemical Marking, Microscopy Handbooks 17, Oxford University Press, 1989, in particular pages 1-14; G. Frens, Nature Physical Science, 241 (1973) 20-22; J. Roth, The Colloidal Gold Marker System for Light and Electron Microscopic Cytochemistry in: Immunocyto-chemistry 2 (1983 218-284)). Explicit reference is made to the disclosure of these documents.
After loading the colloidal particles with the respectively desired biomolecule, it is necessary to stabilize the conjugates. This stabilization is intended to reduce aggregation of the particles and to saturate remaining free surfaces that are accessible to adsorption. Stabilizers used in the state of the art are inert proteins e.g. bovine serum albumin, blood substitute mixtures etc., water-soluble technical polymers such as polyethylene glycol (molecular weight 20,000 D), polyvinylpyrrolidone, polyvinylalcohol, polyvinylsulfate, dextran and gelatin (cf. e.g. De Mey, Supra; Beesley, Supra; Behnke, Eur. J. Cell Biol. 41 (1986), 326-338; DE 24 20 531 C3; and Meisel et al., J. Phys. Chem. 85 (1981), 179-187). In addition the possibility of stabilizing gold sols by phosphane complex ligands has also been described (Schmid et al., Z. Naturforsch. 45b (1994), 989-994).
When loading colloidal gold particles the procedure is usually to adjust the solution of the protein to be adsorbed to the gold as well as the gold sol to a pH close to the isoelectric point (IP) of the protein. In this connection it has been regarded as essential in the state of the art for a successful loading that the protein solution should if possible contain no additives and e.g. the ionic strength should not be above 10 mM. For the loading the protein solution is added to the gold sol while stirring or vice versa. After the protein has bound to the gold particles, a solution of a suitable stabilizer is added. Optionally the conjugate formed is subsequently purified e.g. by ultra-centrifugation or gel filtration.
The stabilizers used according to the state of the art bind adsorptively to the free surfaces of the metal particles. Longer storage or changes in the ambient conditions such as those which occur in a test by contact with the sample (blood, serum, plasma, urine), incorporation of the conjugates in test strip fleeces etc., can desorb or displace the stabilizers from the surfaces to a greater of lesser extent. This leads to a deterioration of the aggregation stability and to an increase in the unspecific reactivity. Moreover most of the stabilizers used are poorly defined products with variable quality in some cases e.g. bovine serum albumin, gelatin. This can also cause variations in the stabilizing effect.
Adsorption processes on particle surfaces are very complex and hitherto are only partially understood. It is assumed that the adsorption is due to a combination of electrostatic interactions, Van-der Waals forces and hydrophobic interactions (Beesley, Supra). Depending on the type of adsorbed biomolecule, the one or the other type of binding can dominate in this process.
Aggregates form to a certain extent in the protein-gold conjugates according to known techniques. These undesired aggregates frequently already occur before addition of the stabilizer. The reasons for the formation of aggregates can for example be that xe2x80x9cstickyxe2x80x9d proteins i.e. proteins with a hydrophobic surface bind together and consequently also bridge the gold particles to which they are conjugated. Therefore it has for example been described that IgG preparations should be freed of aggregates by ultracentrifugation before coupling to gold (W. D. Geoghegan, G. A. Ackerman, J. Histochem. Cytochem. 25 (1977), 1187-1200). Furthermore it is possible that hydrophobic patches that are not covered by protein on the surface of noble medal particles and in particular gold particles can interact with one another and form particle aggregates. A further cause for the occurrence of undesired aggregates can also be that hydrophobic patches on the noble metal surface that are not covered by protein interact with hydrophobic patches on proteins conjugated to neighbouring gold particles and thus cross-link the gold particles.
One frequently also observes a secondary cross-linking of protein gold conjugates which also occurs when the conjugates have already been saturated with a stabilizer. This is probably due to the fact that even after the action of conventional stabilizers, hydrophobic patches remain on the gold surface or/and on the conjugated protein by means of which an uncontrolled aggregation of protein-gold particles occurs with slow kinetics. These problems do not only occur in conjugates containing particles of gold but also in particles made of other solids, especially other metals.
Therefore the object of the present invention was to provide conjugates of colloidal particles and biomolecules in a stable form which do not have the disadvantages of the state of the art.
Surprisingly it has been found that detergents are extremely suitable for stabilizing biomolecule particle conjugates. One subject matter of the invention is therefore a composition which comprises colloidal particles on the surface of which biomolecules are adsorbed wherein the composition additionally contains a detergent.
It was found that the addition of detergents to colloidal particles, for example to a gold sol, before loading with biomolecules and/or to a solution containing biomolecules before and/or during loading of the colloidal particles, impairs binding to the particles to an extent that is considerably less than would have been expected by a person skilled in the art, and is surprisingly advantageous in several respects. The addition of detergent according to the invention prevents aggregation processes that already take place with conventional stabilizers before conjugation and before re-loading and this improves the reproducibility of the manufacturing process for the conjugates and leads to a more uniform size distribution of the conjugates i.e. an increased monodispersity.
A substantial improvement in the stability of the conjugates is achieved compared to the stabilizers of the state of the art. In particular the slow after-aggregation of already stabilized biomolecule particle conjugates which occurs with conventional compositions is suppressed. This leads to an improved long-term stability and a lower aggregation tendency in solution, to an improved stability against changes in the ambient conditions and to an improved test function e.g. improved chromatographic properties. The addition of detergent considerably improves the function of biomolecule-particle conjugates and in particular the function of biomolecule-gold conjugates in tests. Hence for example the non-specific binding which corresponds to the blank reading in the test is reduced.
It was particularly surprising that the improvements could be achieved without adversely influencing the function of the conjugates for example by displacing the biomolecules by the detergent or by possible interactions of the biomolecules or the colloidal particles with the detergent.
The composition according to the invention can be present as an aqueous suspension and also immobilized for example on a chromatographic material such as an absorbent paper.
The particles can be metallic or non-metallic particles such as carbon particles. Metallic particles are preferred such as particles of metals, metal oxides, metal hydroxides, metal compounds or particles coated with metals or metal compounds. Metal particles are particularly preferred.
The metal particles are preferably noble metal particles e.g. metals selected from the group comprising gold, silver, copper, platinum, palladium and mixtures thereof. Gold particles are particularly preferred.
The mean diameter of the particles isxe2x80x94as usual in the state of the artxe2x80x94in the range of 1 to 1000 nm and can be varied according to the application purpose. The mean diameter of the particles is preferably in the range of 2 to 200 nm and especially preferably 2 to 100 nm.
The biomolecules adsorbed to the surface of the particles are preferably selected from the group comprising proteins, glycoproteins, peptides, nucleic acids, peptidic nucleic acids, saccharides, antigens and haptens. Biomolecules are particularly preferably selected from the group comprising antibodies, antibody fragments, lectins, enzymes, streptavidin, avidin, protein A, antigens, such as recombinant polypeptides or multiple antigens (cf. WO96/03652), e.g. polyhaptens (several haptens or peptides coupled to a carrier such as dextran or to a protein), peptides and haptens (low molecular substances preferably with a MW xe2x89xa61500 such as biotin, fluorescein or digoxigenin). With regard to the exact conditions for adsorption of these biomolecules to gold particles, reference is made to the already mentioned review article of De Mey and Beesley.
According to the invention detergents that can be used are anionic, cationic, ampholytic or non-ionic surface-active substances, in particular surfactants. The compositions according to the invention preferably contain an ethoxylate detergent and particularly preferably polyethoxysorbitan laurate and/or oleate or/and laurylpolyethylene glycol ether as a detergent. These detergents are commercially available under the brand names TWEEN(copyright) and Brij(copyright) (e.g. TWEEN 80, TWEEN 20, Brij 35).
The detergent is preferably used at a concentration which does not exceed the critical micelle concentration. In this connection the critical micelle concentration is understood as that concentration at which higher aggregates, so-called micelles, are formed from the surfactant molecules. Attainment of the critical micelle concentration can be easily determined by the jump in physical properties such as for example the surface tension, the osmotic pressure, the equivalent conductivity, the interfacial tension and/or the density. Each of these parameters can be measured with known methods.
The optimal detergent concentration depends on the respective properties of the biomolecule to be conjugated and it must be individually determined for each biomolecule. The optimal detergent concentration is present when, on the one hand, an adequate amount of biomolecules bind to the surface of the colloidal particles and, on the other hand, unspecific hydrophobic interactions are substantially suppressed. The detergent is preferably present at a concentration of 0.0001 to 1 mM, particularly preferably 0.001 to 0.1 mM (final concentration in the conjugation preparation).
The composition according to the invention can be prepared by adding a detergent to colloidal particles before loading with biomolecules or/and to a solution containing biomolecules before or/and during loading of the colloidal particles with this solution.
The compositions according to the invention can be used as a detection reagent, in particular as an immunological detection reagent. In a first preferred embodiment the detection reagent is used in an immunoassay i.e. in a method for the determination of an analyte in a sample liquid by means of immunological methods e.g. by a competitive assay in which a labelled analyte analogue or a labelled analyte-specific receptor e.g. an antibody is used, or a sandwich assay in which a labelled analyte-specific receptor or a labelled additional receptor that is capable of binding to the analyte-specific receptor is used. Preferred examples are pregnancy tests e.g. tests for the detection of human chorionic gonadotrophin (HCG) or methods for the detection of drugs such as cocaine or amphetamines, human serum albumin, troponin T, myoglobin and immunoglobulins such as anti-HIV antibodies. Particularly preferred forms of application are rapid tests in which the sample to be determined is applied to an absorbent material containing the detection reagent e.g. a test strip. A second particularly preferred embodiment in which the stabilized composition according to the invention can be used is the staining of tissue sections.
Furthermore the compositions according to the invention can of course also be used for all further applications that are known for biomolecule particle conjugates e.g. for gene transfer.
Yet a further subject matter of the present invention is a process for stabilizing conjugates of colloidal particles and biomolecules in which a detergent is added to colloidal particles, in particular gold sol, before loading and/or to a solution containing biomolecules before or/and during the loading of the colloidal particles with this solution. In this manner it is possible to achieve an increased long-term stability of the conjugates as well as an improved pH stability and an improved stability against the presence of other substances. In these applications the detergent is preferably used in an amount at which the micelle concentration is not exceeded. The detergent is preferably used in an amount which results in a final concentration of 0.0001 to 1 mM, preferably 0.001 to 0.1 mM.
Furthermore after the loading one can also use additional stabilizers known from the state of the art such as inert proteins e.g. bovine albumin or/and polyethylene glycols.
Finally the present invention also concerns a test kit for an immunological method of detection which contains a composition stabilized according to the invention as a detection reagent.
The invention is further elucidated by the following examples.